Nephrogenic diabetes insipidus (NDI) is a disease characterized by the production of very large quantities of dilute urine from an inability of the kidney to respond to vasopressin. NDI can be either congenital or acquired. Congenital NDI results from mutations in the type 2 vasopressin receptor (V2R) in 90% of families (in which the mutation is known) and in AQP2 in the other 10%. The most common cause of acquired NDI is lithium treatment, a medication that interferes with cAMP signaling. In patients with V2R mutations or taking lithium, there are no mutations in either the AQP2 or UT-A1 proteins, suggesting that if it is possible to phosphorylate these proteins and increase their apical membrane accumulation independent of vasopressin or cAMP, then one may be able to treat, or at least lessen the severity of, the NDI. Therefore, we looked for a kinase that could phosphorylate both AQP2 and UT-A1 independent of vasopressin. Adenosine monophosphate kinase (AMPK) is an energy-sensing kinase that can be stimulated by osmotic stress and hypoxia. AMPK has never been studied in inner medulla, which is normally hypertonic and hypoxic. We have preliminary data showing that AMPK phosphorylates AQP2 and UT-A1, both in vitro and in native rat inner medullary collecting ducts. We also have preliminary data showing that metformin increases urine osmolality in mice lacking a V2R, a mouse model of congenital NDI. In this application, we will test the hypothesis that AMPK, independent of vasopressin, increases urine concentrating ability through increases in AQP2 and UT-A1 phosphorylation, apical plasma membrane accumulation, and function. Specific Aim 1 will test whether AQP2 and UT-A1 are substrates for phosphorylation by AMPK. Specific Aim 2 will test whether phosphorylation of AQP2 and UT-A1 by AMPK increases the apical membrane accumulation of these two proteins. Specific Aim 3 will test whether activation of AMPK increases urea and water permeabilities, and urine concentrating ability in animal models of NDI. If our hypothesis is correct, then AMPK activators would be a novel therapeutic option for congenital NDI due to V2R mutations, and for acquired forms of NDI due to interference with cAMP.